Labeling of peroxisomes with green fluorescent protein in living P-pastoris cells
Labeling of peroxisomes with green fluorescent protein in living P-pastoris cells
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DOI：
10.1177/44.6.8666743
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发表时间：
1996-06-01
影响因子：
3.2
通讯作者：
Subramani, S
中科院分区：
文献类型：
--
作者：
Monosov, EZ;Wenzel, TJ;Subramani, S
我们利用水母绿色荧光蛋白(GFP)的光激活荧光特性来研究多肽的过氧化体分选，GFP和GFP-SKL(含有一个C末端，三肽过氧化体靶向信号，SKL)是在甲醇诱导的乙醇氧化酶(AOX1)启动子的甲醇营养酵母巴斯德酵母中表达的。Nycodenz梯度上细胞器的生化分级表明，GFP和GFP-SKL融合蛋白是胞浆的，而GFP-SKL融合蛋白针对的是过氧化体，GFP和GFP-SKL对酵母细胞的活力没有影响，但在395 nm紫外光激发下，两者都是荧光的。通过荧光显微镜观察GFP和GFP-SKL在活酵母细胞中的亚细胞位置，并将它们的荧光与二氨基联苯胺(DAB)的光氧化偶联，导致电子致密的氧化DAB沉积在GFP衍生物的胞内位置。这种光氧化过程允许通过电子显微镜对GFP在细胞内进行简单的超微结构定位，并提供了进一步的证据，证明在巴斯德毕赤酵母中产生的GFP是胞质的，而GFP-SKL是过氧体。因此，GFP-SKL融合蛋白是过氧体隔室的通用报告蛋白，在涉及过氧体输入和生物发生的研究中有许多应用。
We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides, GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients, Neither GFP nor GFP-SKL affected the viability of yeast cells bur both were fluorescent on excitation with 395-nm uv light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives, This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL, is peroxisomal, The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.